Single-cell characterization of the eukaryotic transcription cycle using live imaging and statistical inference

AbstractThe eukaryotic transcription cycle consists of three main steps: initiation, elongation, and cleavage of the nascent RNA transcript. Although each of these steps can be regulated as well as coupled with each other, their in vivo dissection has remained challenging because available experimental readouts lack sufficient spatiotemporal resolution to separate the contributions from each of these steps. Here, we describe a novel computational technique to simultaneously infer the effective parameters of the transcription cycle in real time and at the single-cell level using a two-color MS2/PP7 reporter gene and the developing fruit fly embryo as a case study. Our method enables detailed investigations into cell-to-cell variability in transcription-cycle parameters with high precision. These measurements, combined with theoretical modeling, reveal a significant variability in the elongation rate of individual RNA polymerase molecules. We further illustrate the power of this technique by uncovering a novel mechanistic connection between RNA polymerase density and nascent RNA cleavage efficiency. Thus, our approach makes it possible to shed light on the regulatory mechanisms in play during each step of the transcription cycle in individual, living cells at high spatiotemporal resolution.

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Real-time single-cell characterization of the eukaryotic transcription cycle reveals correlations between RNA initiation, elongation, and cleavage

PLoS Computational Biology ◽

10.1371/journal.pcbi.1008999 ◽

2021 ◽

Vol 17 (5) ◽

pp. e1008999

Author(s):

Jonathan Liu ◽

Donald Hansen ◽

Elizabeth Eck ◽

Yang Joon Kim ◽

Meghan Turner ◽

...

Keyword(s):

Rna Polymerase ◽

Single Cell ◽

Real Time ◽

Fruit Fly ◽

Effective Parameters ◽

Spatiotemporal Resolution ◽

Eukaryotic Transcription ◽

Nascent Rna ◽

Transcription Cycle

The eukaryotic transcription cycle consists of three main steps: initiation, elongation, and cleavage of the nascent RNA transcript. Although each of these steps can be regulated as well as coupled with each other, their in vivo dissection has remained challenging because available experimental readouts lack sufficient spatiotemporal resolution to separate the contributions from each of these steps. Here, we describe a novel application of Bayesian inference techniques to simultaneously infer the effective parameters of the transcription cycle in real time and at the single-cell level using a two-color MS2/PP7 reporter gene and the developing fruit fly embryo as a case study. Our method enables detailed investigations into cell-to-cell variability in transcription-cycle parameters as well as single-cell correlations between these parameters. These measurements, combined with theoretical modeling, suggest a substantial variability in the elongation rate of individual RNA polymerase molecules. We further illustrate the power of this technique by uncovering a novel mechanistic connection between RNA polymerase density and nascent RNA cleavage efficiency. Thus, our approach makes it possible to shed light on the regulatory mechanisms in play during each step of the transcription cycle in individual, living cells at high spatiotemporal resolution.

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Transcriptional Pausing in Vivo: A Nascent RNA Hairpin Restricts Lateral Movements of RNA Polymerase in Both Forward and Reverse Directions

Journal of Molecular Biology ◽

10.1016/j.jmb.2005.05.052 ◽

2005 ◽

Vol 351 (1) ◽

pp. 39-51 ◽

Cited By ~ 18

Author(s):

Francine Toulmé ◽

Christine Mosrin-Huaman ◽

Irina Artsimovitch ◽

A. Rachid Rahmouni

Keyword(s):

Rna Polymerase ◽

Transcriptional Pausing ◽

Nascent Rna ◽

Rna Hairpin

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Differential Responses of Transplanted Stem Cells to Diseased Environment Unveiled by a Molecular NIR-II Cell Tracker

Research ◽

10.34133/2021/9798580 ◽

2021 ◽

Vol 2021 ◽

pp. 1-16

Author(s):

Hao Chen ◽

Huaxiao Yang ◽

Chen Zhang ◽

Si Chen ◽

Xin Zhao ◽

...

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Cell Therapy ◽

Single Cell ◽

Real Time ◽

Stem Cell Therapy ◽

Cellular Migration ◽

Cell Cluster ◽

Spatiotemporal Resolution

Stem cell therapy holds high promises in regenerative medicine. The major challenge of clinical translation is to precisely and quantitatively evaluate the in vivo cell distribution, migration, and engraftment, which cannot be easily achieved by current techniques. To address this issue, for the first time, we have developed a molecular cell tracker with a strong fluorescence signal in the second near-infrared (NIR-II) window (1,000-1,700 nm) for real-time monitoring of in vivo cell behaviors in both healthy and diseased animal models. The NIR-II tracker (CelTrac1000) has shown complete cell labeling with low cytotoxicity and profound long-term tracking ability for 30 days in high spatiotemporal resolution for semiquantification of the biodistribution of transplanted stem cells. Taking advantage of the unique merits of CelTrac1000, the responses of transplanted stem cells to different diseased environments have been discriminated and unveiled. Furthermore, we also demonstrate CelTrac1000 as a universal and effective technique for ultrafast real-time tracking of the cellular migration and distribution in a 100 μm single-cell cluster spatial resolution, along with the lung contraction and heart beating. As such, this NIR-II tracker will shift the optical cell tracking into a single-cell cluster and millisecond temporal resolution for better evaluating and understanding stem cell therapy, affording optimal doses and efficacy.

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Transcription of fractionated mammalian chromatin by mammalian ribonucleic acid polymerase. Demonstration of temperature-dependent rifampicin-resistant initiation sites in euchromatin deoxyribonucleic acid

Biochemical Journal ◽

10.1042/bj1430073 ◽

1974 ◽

Vol 143 (1) ◽

pp. 73-81 ◽

Cited By ~ 11

Author(s):

C. James Chesterton ◽

Barbara E. H. Coupar ◽

Peter H. W. Butterworth

Keyword(s):

Rna Polymerase ◽

Rat Liver ◽

Deoxyribonucleic Acid ◽

Temperature Dependent ◽

Fractionation Method ◽

Liver Nuclei ◽

Nascent Rna ◽

Chromatin Fractionation

The chromatin fractionation method of Frenster et al. (1963) as modified by Leake et al. (1972) was used to prepare fragments of euchromatin from rat liver nuclei. These remain soluble in 5mm-MgCl2, and contain DNA of maximum mol.wt. 1×106–2×106. The fragments were separated from condensable chromatin on a sucrose gradient. Euchromatin contains endogenous DNA-dependent RNA polymerase, and most of the nascent RNA labelled in vivo or in vitro. Euchromatin fragments allow initiation of transcription by added purified rat liver form-B RNA polymerase and contain temperature-dependent rifampicin-resistant initiation sites for the form-B enzyme. These findings indicate that transcription of the euchromatin regions of interphase chromosomes is not initiated in condensed chromatin, but is initiated within the euchromatin stretches. Condensable chromatin also contains most of these activities, but is not associated with nascent RNA.

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Role for the Ssu72 C-Terminal Domain Phosphatase in RNA Polymerase II Transcription Elongation

Molecular and Cellular Biology ◽

10.1128/mcb.01361-06 ◽

2006 ◽

Vol 27 (3) ◽

pp. 926-936 ◽

Cited By ~ 39

Author(s):

Mariela Reyes-Reyes ◽

Michael Hampsey

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Transcription Elongation ◽

Elongation Factor ◽

Elongation Complex ◽

Terminal Domain ◽

Rnap Ii ◽

Transcription Cycle

ABSTRACT The RNA polymerase II (RNAP II) transcription cycle is accompanied by changes in the phosphorylation status of the C-terminal domain (CTD), a reiterated heptapeptide sequence (Y1S2P3T4S5P6S7) present at the C terminus of the largest RNAP II subunit. One of the enzymes involved in this process is Ssu72, a CTD phosphatase with specificity for serine-5-P. Here we report that the ssu72-2-encoded Ssu72-R129A protein is catalytically impaired in vitro and that the ssu72-2 mutant accumulates the serine-5-P form of RNAP II in vivo. An in vitro transcription system derived from the ssu72-2 mutant exhibits impaired elongation efficiency. Mutations in RPB1 and RPB2, the genes encoding the two largest subunits of RNAP II, were identified as suppressors of ssu72-2. The rpb1-1001 suppressor encodes an R1281A replacement, whereas rpb2-1001 encodes an R983G replacement. This information led us to identify the previously defined rpb2-4 and rpb2-10 alleles, which encode catalytically slow forms of RNAP II, as additional suppressors of ssu72-2. Furthermore, deletion of SPT4, which encodes a subunit of the Spt4-Spt5 early elongation complex, also suppresses ssu72-2, whereas the spt5-242 allele is suppressed by rpb2-1001. These results define Ssu72 as a transcription elongation factor. We propose a model in which Ssu72 catalyzes serine-5-P dephosphorylation subsequent to addition of the 7-methylguanosine cap on pre-mRNA in a manner that facilitates the RNAP II transition into the elongation stage of the transcription cycle.

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In vivo NIR-II structured-illumination light-sheet microscopy

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2023888118 ◽

2021 ◽

Vol 118 (6) ◽

pp. e2023888118

Author(s):

Feifei Wang ◽

Zhuoran Ma ◽

Yeteng Zhong ◽

Felix Salazar ◽

Chun Xu ◽

...

Keyword(s):

Light Scattering ◽

Molecular Imaging ◽

Single Cell ◽

Light Sheet ◽

Single Cell Level ◽

Structured Illumination ◽

Cell Level ◽

Spatiotemporal Resolution ◽

Light Sheet Microscopy

Noninvasive optical imaging with deep tissue penetration depth and high spatiotemporal resolution is important to longitudinally studying the biology at the single-cell level in live mammals, but has been challenging due to light scattering. Here, we developed near-infrared II (NIR-II) (1,000 to 1,700 nm) structured-illumination light-sheet microscopy (NIR-II SIM) with ultralong excitation and emission wavelengths up to ∼1,540 and ∼1,700 nm, respectively, suppressing light scattering to afford large volumetric three-dimensional (3D) imaging of tissues with deep-axial penetration depths. Integrating structured illumination into NIR-II light-sheet microscopy further diminished background and improved spatial resolution by approximately twofold. In vivo oblique NIR-II SIM was performed noninvasively for 3D volumetric multiplexed molecular imaging of the CT26 tumor microenvironment in mice, longitudinally mapping out CD4, CD8, and OX40 at the single-cell level in response to immunotherapy by cytosine-phosphate-guanine (CpG), a Toll-like receptor 9 (TLR-9) agonist combined with OX40 antibody treatment. NIR-II SIM affords an additional tool for noninvasive volumetric molecular imaging of immune cells in live mammals.

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Fluorescent labeling of nascent RNA reveals transcription by RNA polymerase II in domains scattered throughout the nucleus

The Journal of Cell Biology ◽

10.1083/jcb.122.2.283 ◽

1993 ◽

Vol 122 (2) ◽

pp. 283-293 ◽

Cited By ~ 327

Author(s):

DG Wansink ◽

W Schul ◽

I van der Kraan ◽

B van Steensel ◽

R van Driel ◽

...

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Single Gene ◽

Fluorescent Labeling ◽

Double Labeling ◽

Preferential Localization ◽

Nascent Rna ◽

Nuclear Domains

Several nuclear activities and components are concentrated in discrete nuclear compartments. To understand the functional significance of nuclear compartmentalization, knowledge on the spatial distribution of transcriptionally active chromatin is essential. We have examined the distribution of sites of transcription by RNA polymerase II (RPII) by labeling nascent RNA with 5-bromouridine 5'-triphosphate, in vitro and in vivo. Nascent RPII transcripts were found in over 100 defined areas, scattered throughout the nucleoplasm. No preferential localization was observed in either the nuclear interior or the periphery. Each transcription site may represent the activity of a single gene or, considering the number of active pre-mRNA genes in a cell, of a cluster of active genes. The relation between the distribution of nascent RPII transcripts and that of the essential splicing factor SC-35 was investigated in double labeling experiments. Antibodies against SC-35 recognize a number of well-defined, intensely labeled nuclear domains, in addition to labeling of more diffuse areas between these domains (Spector, D. L., X. -D. Fu, and T. Maniatis. 1991. EMBO (Eur. Mol. Biol. Organ.) J. 10:3467-3481). We observe no correlation between intensely labeled SC-35 domains and sites of pre-mRNA synthesis. However, many sites of RPII synthesis colocalize with weakly stained areas. This implies that contranscriptional splicing takes place in these weakly stained areas. These areas may also be sites where splicing is completed posttranscriptionally. Intensely labeled SC-35 domains may function as sites for assembly, storage, or regeneration of splicing components, or as compartments for degradation of introns.

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